IL-35 is a novel responsive anti-inflammatory cytokine--a new system of categorizing anti-inflammatory cytokines.

1Department of Pharmacology and Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, United States of America.

Abstract

It remains unknown whether newly identified anti-inflammatory/immunosuppressive cytokine interleukin-35 (IL-35) is different from other anti-inflammatory cytokines such as IL-10 and transforming growth factor (TGF)-β in terms of inhibition of inflammation initiation and suppression of full-blown inflammation. Using experimental database mining and statistical analysis methods we developed, we examined the tissue expression profiles and regulatory mechanisms of IL-35 in comparison to other anti-inflammatory cytokines. Our results suggest that in contrast to TGF-β, IL-35 is not constitutively expressed in human tissues but it is inducible in response to inflammatory stimuli. We also provide structural evidence that AU-rich element (ARE) binding proteins and microRNAs target IL-35 subunit transcripts, by which IL-35 may achieve non-constitutive expression status. Furthermore, we propose a new system to categorize anti-inflammatory cytokines into two groups: (1) the house-keeping cytokines, such as TGF-β, inhibit the initiation of inflammation whereas (2) the responsive cytokines including IL-35 suppress inflammation in full-blown stage. Our in-depth analyses of molecular events that regulate the production of IL-35 as well as the new categorization system of anti-inflammatory cytokines are important for the design of new strategies of immune therapies.

A. Data presentation format (The data presented in X-, Y-axis, and tissue order of ARHGDIA and Idha are applied to all the human and mouse genes examined respectively). As an example, the gene expression profiles of human housekeeping gene Rho GDP dissociation inhibitor (GDI) alpha (ARHGDIA) in the eighteen tissues are presented, with the tissue names and position numbers shown on the X-axis. The gene expression data were normalized by the β-actin (Hs. 520640) expression data from the same tissue, which are presented on the left Y-axis. The expression ratios among tissues were generated by normalizing the arbitrary units of the gene in the tissues with the median level of the arbitrary units of the gene in all the tissues which are presented on the right Y-axis. In order to define confidence intervals for statistically higher expression levels of given genes, we calculated the confidence intervals of tissue expression for three housekeeping genes [the mean X+2×standard deviations (SD) = 2.83] including ARHGDIA (Hs. 159161), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs. 544577), and ribosomal protein S27a (RPS27A, Hs. 311640). The expression variations of given genes in tissues, when they were larger than 2.83-fold, were defined as the high expression levels with statistical significance (the right Y-axis). To define confidence intervals for statistically higher expression levels of given genes in 14 mouse tissues, we calculated the confidence intervals of tissue expression [the mean X+2×standard deviations (SD) = 3.1] for three mouse house keeping genes including Lactate dehydrogenase A (Ldha, Mm. 29324), non-POU-domain-containing octamer binding protein (Nono, Mm. 280069), and ribosomal protein L32 (Rpl32, Mm. 104368). The expression variations of given genes in tissues, when they were larger than 3.1-fold, were defined as the high expression levels with statistical significance (the right Y-axis). B. The expression profiles of suppressive cytokines in human (Left 2 columns, with cytokine members designated with capital letters) and mouse (right 2 columns, with cytokine members designated with lowercase letters) tissues. C. The expression profiles of IL-35 related subunits in human and mouse tissue.

Transcription factor binding frequencies in the promoter region of IL-35.

The promoter sequences (1500 base pair upstream of the transcription start site) of 3 housekeeping genes (ACTB, GAPDH, ARHGDIA), IL12A, EBI3, and TGFB1 were retrieved from the NIH/NCBI Entrez Gene database, and were analyzed using TESS to determine the frequencies of 10 Transcription factors (TFs). The binding frequencies of the 10 TFs in the promoter region of each gene were counted. Confidence interval was set by using the mean+2×standard deviation (SD) of the TF binding frequencies in the promoter of 3 housekeeping genes. The binding frequency which is higher than the uppermost confidence interval (p<0.05) is considered significant.

A. Concentrations of SAM and SAH in mouse tissues were previously examined by Ueland et al. B. Correlation of suppressive cytokines and TGF-β receptors with SAM/SAH ratios in mouse tissues. C. Schematic presentation of how IL-35 may be regulated by methylation status. S-Adenosylhomocysteine (SAH) and S-Adenosylmethionine (SAM) are intermediate metabolites of homocysteine-methionine metabolism cycle. SAH is a potent inhibitor of cellular methylation. High SAM/SAH ratio is associated with hypermethylation of DNA and no IL12A/Ebi3 expression. Low SAM/SAH ratio is associated with hypomethylation of DNA and Ebi3 can be expressed.